Noboru Ototani

Purification of chondroitinase B and chondroitinase C using glycosaminoglycan-bound AH-Sepharose 4B.

Chondroitinase B and chondroitinase C were separated from an extract of Flavobacterium heparinum induced with chondroitin 6-sulfate by using column chromatography on hydroxylapatite. Chondroitinase C was eluted together with the activities of hyaluronidase, delta4,5glycosiduronase, and sulfatase. The latter two activities were eliminated exclusively by passing the crude chondroitinase C fraction through a phosphono-cellulose column pre-equilibrated with 0.07M sodium phosphate buffer (pH 6.8). Chondroitinase C was then purified by affinity chromatography using dermatan sulfate-bound AH-Sepharose 4B coated with the same glycosaminoglycan. Purification of the enzyme was achieved 18-fold and in 73% yield. On the other hand, the activities of delta4,5glycosiduronase and sulfatase were decreased to 50 and 60%, respectively, as compared with those in the crude chondroitinase B fraction, after passing the fraction through a column of phosphono-cellulose pre-equilibrated with 0.1M sodium phosphate buffer (pH 6.8). The remaining activities of these two enzymes were then eliminated from chondroitinase B by affinity chromatography with heparin-bound AH-Sepharose 4B coated with dermatan sulfate. In the affinity chromatography used in the present study, non-covalent coating of the glycosaminoglycan-bound (covalently) AH-Sepharose 4B with the same or another glycosaminoglycan was found to be important.

Isolation and characterization of chondroitin sulfate proteoglycans from porcine thoracic aorta

A chondroitin sulfate proteoglycan fraction was prepared from the 3 M MgCl2 extract of porcine aortas by DEAE-cellulose chromatography, followed by gel filtration through Sepharose CL-4B. Affinity chromatography of the fraction with antithrombin III-agarose yielded two chondroitin sulfate proteoglycans of a non-binding (proteoglycan IA) and binding (proteoglycan IB) nature. Proteoglycans IA and IB were different from each other in molecular size, in proportion of the protein relative to the polysaccharide portion, and in size of the chondroitin sulfate chain. They were also distinguished immunochemically. These data indicate that the intima-media of the aorta contains at least two distinct species of chondroitin sulfate proteoglycan.

Isolation of dermatan sulfate with high heparin cofactor II-mediated thrombin-inhibitory activity from porcine spleen

We prepared dermatan sulfate specimens from various porcine tissues, and compared their heparin cofactor II-mediated thrombin-inhibitory activities and chemical natures, including disaccharide composition. Electrophoresis of the specimens on cellulose acetate membrane indicated that spleen dermatan sulfate was the most acidic of the dermatan sulfates prepared from the various porcine tissues. Analysis of the disaccharide units of the dermatan sulfate specimens by high-performance liquid chromatography revealed that spleen dermatan sulfate was rich in 4,6-di-O-sulfated N-acetylgalactosamine residues as compared with those of the other tissues. Spleen dermatan sulfate exhibited the highest thrombin-inhibitory activity, which may be related to its high content of the disulfated N-acetylgalactosamine residue.

A simple method for the quantitation of glycuronic acid-containing glycosaminoglycans with mucopolysaccharidases

A simple method for the quantitative determination of glycuronic acid-containing glycosaminoglycans (UA-GAG) is described. Sample solutions of glycosaminoglycans were digested with chondroitinase AC, chondroitinase C, chondroitinase B, heparitinases, and Streptomyces hyaluronidase, respectively, and the absorbance was read at 232 nm after digestion. The contents of 4-O-sulfated N-acetylgalactosaminyl beta (1 leads to 4)D-glucosiduronyl units (Ch-4S), 6-O-sulfated N-acetylgalactosaminyl beta (1 leads to 4)D-glucosiduronyl units (Ch-6S) plus N-acetylgalactosaminyl beta (1 leads to 4)D-glucosiduronyl units (Ch-OS), 4-O-sulfated N-acetylgalactosaminyl beta (1 leads to 4)L-idosiduronyl units (D-4S) plus N-acetylgalactosaminyl beta (1 leads to 4)L-idosiduronyl units (D-OS), heparan sulfate, and hyaluronic acid in the sample solutions were calculated from the absorbance with reference to that of the digestion products of known amounts of standard UA-GAG. The analytical data obtained with the mixtures of authentic UA-GAG were in close agreement with the theoretical values. Application of this procedure to the urinary GAG fractions from orthopedic patients gave satisfactory results.

Preparation of phenyl 4-deoxy-α- and β-l-threo-hex-4-enopyranosiduronic acids and determination of the anomeric specificity of the Δ4,5-glycosiduronase induced from flavobacterium heparinum with heparin and chondroitin sulfate

Abstract To investigate the anomeric specificity of the Δ4,5-glycosiduronase induced from Flavobacterium heparinum with heparin and chondroitin sulfate, phenyl 4-deoxy-α- and β- l -threo-hex-4-enopyranosiduronic acids were chemically synthesized and then digested with the purified enzymes. Only the α- l anomer of the unsaturated uronic acid was degraded by the purified enzymes induced from the microbe with heparin or chondroitin sulfate. It was also confirmed that the Δ4,5-glycosiduronase induced with heparin and that induced with chondroitin sulfate hydrolyzed exclusively 2-acetamido-2-deoxy-4-O-(4-deoxy-α- l -threo-hex-4-enopyranosyluronic acid)- d -glucose and 2-acetamido-2-deoxy-3-O-(4-deoxy-α- l -threo-hex-4-enopyranosyluronic acid)- d -galactose, respectively. It was concluded that the purified preparation of Δ4,5-glycosiduronase induced with heparin was specific for the (1→4)-α- l -threo-4-enopyranosyluronic acid linkage, whereas that of the Δ4,5-glycosiduronase induced with chondroitin sulfate was specific for the (1→3)-α- l -threo-4-enopyranosyluronic acid linkage.

A typical dermatan sulfate isolated from whale intestine

Alkaline extraction of whale intestine, followed by pronase digestion and precipitation of heparin (omega-heparin) with dodecyltrimethylammonium chloride gave a supernatant fraction containing dermatan sulfate. Ethanol at 20% concentration precipitated dermatan sulfate from the supernatant fraction. The crude dermatan sulfate was further fractionated by ion-exchange column chromatography on Dowex-1 (C1- form), eluting stepwise with aqueous sodium chloride. The fractions eluted with 1.5M and 1.75M sodium chloride contained a typical dermatan sulfate. Chemical and enzymic studies of these preparations revealed that the sulfate groups were located solely at O-4 of the 2-acetamido-2-deoxy-D-galactose resides. L-Iduronic acid was assumed to be distributed uniformly in the backbone of the polysaccharide chain, with D-glucuronic acid being located in the linkage region to the protein core. A new method for determining the ratio of D-glucuronic to L-iduronic acid is also described.

Ether cleavage of 7-oxabicyclo[2,2,1]heptane derivatives

In connection with the total synthesis of natural products containing a 2-hydroxycyclohexane-carboxylic acid or -1,5-carbolactone system, ether cleavage of 1-methyl-7-oxabicyclo[2,2,1]heptane-2,3-dicarboxylic acids and their methyl esters was achieved. The products included 6-acetoxy-3-methylcyclohex-3-ene-1,2-dicarboxylic anhydride, the corresponding dicarboxylic acids and cis-dimethyl ester, 1-methylcyclohexane-2,4:3,1-biscarbolactone, 6-acetoxy-2-carboxy-3-methylcyclohexane-1,3-carbolactone, and dimethyl 3,6-diacetoxy-3-methylcyclohexane-1,2-dicarboxylate.

An approach to the synthesis of fujenoic acid

In experiments directed towards the total synthesis of fujenoic acid (1), the 2-(2-furyl)-6-oxobicyclo[3.2.1]octane-1-carboxylate (3b) was prepared as a key intermediate. The furyl group was designed to be transformed into ring A of fujenoic acid. In investigations of the transformation route, model compounds {the stereoisomeric dimethyl 1-methyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylates (4), (5), and (12)} were examined, and it was found that diester (12) could be effectively converted into dimethyl 1,3-dimethyl-6-oxocyclohex-3-ene-1,2-dicarboxylate (18), which corresponds to ring A of fujenoic acid. The key intermediate (3b) was obtained by a Dieckmann condensation of dimethyl 6-(2-furyl)-1-methoxycarbonylmethylcyclohexane-1,3-dicarboxylate (23).